1. Field of the Invention
The present invention provides chimeric polypeptides having one or more glutamic acid decarboxylase (GAD) 65 epitopes, wherein the chimeric polypeptides can specifically bind a GAD antibody present in serum from a subject having insulin-dependent diabetes mellitus (IDDM, also referred to as type I diabetes). The chimeric polypeptides can be used to detect individuals having new-onset diabetes, who are in the prediabetic stage of type I diabetes or to classify the type of diabetes in an individual as type I.
2. Background Art
Insulin-dependent diabetes mellitus (IDDM) (also known as type 1 diabetes) primarily afflicts young people. Although insulin is available for treatment, the several-fold increased morbidity and mortality associated with this disease require the development of early diagnostic and preventive methods. The destruction of pancreatic .beta.-cells (which are the insulin-secreting cells of the islets of Langerhans) that precedes the clinical onset of IDDM, is mediated by autoimnuune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high incidence of circulating .beta.-cell specific autoantibodies at the time of diagnosis. Family studies have shown that the autoantibodies appear prior to overt IDDM by a number of years, suggesting a long prodromal period of humoral autoimmunity before clinical symptoms emerge. The family studies have also documented a slow, progressive loss of insulin response to intravenous glucose in the years preceding diagnosis. The presence of .beta.-cell specific autoantibodies in the prediabetic period is likely to reflect the ongoing autoimmune process, one that eventually leads to critical .beta.-cell depletion and insulin dependency. It has been estimated that only 10% of the total .beta.-cell mass remains at the time of clinical onset.
A major autoantigen for both humoral and cellular autoimmmunity in insulin-dependent diabetes mellitus (EDM) is glutamic acid decarboxylase (GAD) (Baekkeskov et al. (1990), Nature 347:151-156; Solimena (1991), Trends in Neurosciences 14:452-457; Clare-Salzler et al. (1992), Diabetes Care 15:132-135). The possible role of GAD as the primary autoantigen in IDDM has been suggested by recent demonstrations of cellular autoimmunity directed at GAD in IDDM (Honeyman et al. (1993), J. Exp. Med. 177:535-540; Atkinson et al. (1992), Lancet 339:458-459) as well as autoimmunity directed at GAD in an animal model of IDDM (Kaufman et al. (1993), Nature 366:69-72; Tisch et al. (1993), Nature 366:72-75). GAD autoantibodies are an important marker of the autoimmune process of IDDM since they are present in the majority of individuals with new-onset diabetes and in individuals in the pre-diabetic stage of the disease (Hagopian et al. (1993), J. Clin. Invest. 91:368-374; Rowley et al. (1992) Diabetes 41:548-551; De Aizpurua et al. (1992), Proc. Natl. Acad. Sci. USA 89:9841-9845).
GAD is the biosynthetic enzyme for the inhibitory neurotransritter .gamma.-amino butyric acid (Erlander et al. (1991), Neurochem. Res. 16:215-226). Two isoforms, GAD.sub.65 (65 kDa) and GAD.sub.67 (67 kDa) are the products of two separate genes, are highly homologous, and differ mostly in the amino terminal-third of the protein (Erlander et al. (1991); Bu et al. (1992), Proc. Natl. Acad. Sci. USA 89:2115-2119; Christgau et al. (1991), J. Biol. Chem. 266:21257-21269). The respective cDNAs for GAD.sub.65 and GAD.sub.67 are identical in the brain and islet (Clare-Salzer et al. (1992), Diabetes Care 15:132-135; Giorda et al. (1991), Lancet 338:1469-1470; Kelly et al. (1991), Lancet 338:1468-1469; Karlsen et al. (1991), Proc. Nat. Acad. Sci. USA 88:8337-8341; Cram et al. (1991), Biochem. Biophys. Res. Commun. 176:1239-1244; Michelson et al. (1991), Proc. Natl. Acad. Sci. USA 88:8754-8758). In the human, GAD.sub.65 is the predominant form expressed in the islet, whereas both GAD.sub.65 and GAD.sub.67 are found in rat islets (Velloso et al. (1993), Diabetologia 36:39-46; Kim et al. (1993), Diabetes 42:1799-1808). Mouse islets contain little, if any, GAD.sub.65 (Velloso et al. (1993); Kim et al. (1993).
Antibodies against GAD, originally detected by Baekkeskov et al. (1993), are found in the majority of individuals with preclinical and new-onset type I diabetes and have been detected utilizing recombinant GAD and porcine brain GAD (Velloso et al. (1993) J. Clin. Invest. 91:2084-2090; Hagopian et al. (1993) J. Clin. Invest. 91:368-374; Karlsen et al. (1992) Diabetes 41:1355-1359; Christie et al. (1992) Diabetologia 35:380-384; Rowley et al. (1992), Diabetes 41:548-551; Clare-Salzler et al. (1992), Diabetes Care 15:132-135; DeAizpurua et al. (1992), Diabetes 41:1182-1187; DeAizpurua et al. (1992), Proc. Natl. Acad. Sci. USA 89:9841-9845). Some ICA sera react with GAD.sub.65 and GAD.sub.67, but GAD.sub.65 is the predominant autoantigen (Velloso et al. (1993); Hagopian et al. (1993), Diabetes 42:631-636). An initial report mapped the reactivity of four ICA sera to the middle and COOH-terminal of GAD.sub.65 Kaufman et al. (1992) J. Clin. Invest. 89:283-292). A recent study using a series of GAD-directed monoclonal antibodies from a single patient with type I diabetes has detected at least two distinct epitopes, one that is linear in nature and one that is dependent on protein conformation (Richter et al. (1993), Proc. Natl. Acad. Sci. USA 90:2832-2836; Richter et al. (1992), Proc. Natl. Acad. Sci. USA 89:8467-8471).
GAD.sub.67 and GAD.sub.65 are highly diverse in the first 95 amino acids but share significant (approx. 75%) homology in the rest of the molecule. Both have a proteolytic hot spot 80-90 amino acids from the N-terminus (Christgau et al. (1991), J. Biol. Chem. 266:21257-21264; Christgau et al. (1992), J. Cell Biol. 118:309-320) (incorporated by reference in their entirety for all purposes), which may represent a domain boundary. The N-terminal domain harbors the post-translational modifications which result in anchoring of GAD.sub.65 to the membrane of synaptic vesicles and control the distinct subcellular localization of this protein.
In brain tissue, both GAD.sub.65 and GAD.sub.67 are produced (Bu et al. (1992), Proc. Natl. Acad. Sci. USA 89:2115-2119; Kaufman et al. (1986) Science 232:1138-1140; Chang & Gottlieb (1988), J. Neurosci. 8:2123-2130). Some species express both GAD proteins in their pancreatic islets. However, in human islets only GAD.sub.65 is expressed (Karlsen et al. (1991), Proc. Natl. Acad. Sci. (USA) 88:8337-8341; Karlsen et al. (1992), Diabetes 41:1355-1359). Immunogenic crossreactivity between isolates of GAD.sub.65 and GAD.sub.67 from different vertebrate species indicates a high degree of conservation of antigenic determinants from rodents to humans (Legay et al. (1986), J. Neurochem. 46:1478-1486). Consistent with this observation, human GAD.sub.65 and GAD.sub.67 polypeptides share more than 80% amino-acid sequence identity with cognate polypeptides in other mammals. Bu et al., supra.
GAD autoantibodies that bind the intact, full length GAD65 protein are also found in some individuals without IDDM including individuals with stiff-man syndrome (SMS), polyglandular failure type I, and individuals who have a restricted or beta-cell specific islet cell autoantibody (ICA) pattern (Solimena et al. (1991), Trends in Neurosciences 14:452-457; Genovese et al. (1992), Diabetologia 35:385-388; Gianani et al. (1992), Diabetes 41:347-353; Bjork et al. (1994), Diabetes 43:161-165). These individuals have a low risk of developing IDDM. Differences and heterogeneity in the GAD autoantibody profile between these non-diabetic individuals and those with IDDM suggest that disease-specific epitopes may exist (Bjork et al. (1994), Diabetes 43:161-165; Butler et al. (1993), J. Exp. Med. 178:2097-2106; Kim et al. (1994), J. Exp. Med. 180:595-606; Ujihara et al. (1994), Diabetes 43:968-975). For example, SMS sera bind GAD protein fagments and denatured GAD protein utilized in immunoblotting (Baekkeskov et al. (1990), Nature 347:151-156; Butler et al. (1993), J. Exp. Med. 178:2097-2106; Kim et al. (1994). J Exp. Med. 180:595-606).
In contrast, GAD antibodies in IDDM do not bind denatured GAD protein, GAD fragments, or GAD peptides which implies that these GAD antibodies bind an epitope dependent on protein conformation (Baekkeskov et al. (1990), Nature 347:151-156; Ujihara et al. (1994), Diabetes 43:968-975; Richter et al. (1993), Proc Natl Acad Sci USA 90:2832-2836). The COOH-terminal two-thirds of the GAD 65 protein contains the region targeted in IDDM, but when smaller fagments of GAD 65 protein are used, most binding by IDDM sera is lost (Ujihara et al. (1994), Diabetes 43:968-975; Richter et al. (1993), Proc Natl Acad Sci USA 90:2832-2836). Hence, further localization of the IDDM epitopes is not possible with the methodology used to map GAD epitopes targeted by SMS or restricted ICA sera. Furthermore, when native, full length GAD65 is used, some sera from individuals without IDDM who have, for example, SMS, polyglandular renal failure type I, or a restricted or .beta.cell-specific ICA pattern bind the GAD65 protein causing false positives for diagnosis of IDDM.
The present invention overcomes these experimental limitations in localizing IDDM-related GAD epitopes by designing recombinant GAD proteins that maintain the conformation of the GAD protein. By exchanging regions of the GAD 65 and GAD 67 cDNAs, we created chimeric GAD polypeptides and determined the binding of IDDM antibodies to different regions of the GAD 65 protein. These chimeric polypeptides utilize one or more epitopes of GAD 65 such that the chimeric protein can bind IDDM sera. The chimeric polypeptides of the present invention provide more specific binding for IDDM GAD autoantibodies than intact GAD65, and produce fewer false positives for binding of IDDM-specific GAD autoantibodies and, thus, provide a more useful diagnostic polypeptide.